1. Technical Field
The present invention relates to methods for detecting the effects of selected growth factors and pharmaceuticals on hair follicle cell proliferation and cellular collagenolytic factor secretion.
2. Background Information
Hair follicle morphogenesis in developing skin is a complex process involving induction of endophytic epidermal buds by underlying mesenchyme, and proliferation, migration, and differentiation of the epithelial component to form a three dimensional structure spanning the deep dermis to the epidermal surface. The activity of this organ cycles throughout the lifetime of the animal as the active (anagen) hair follicle regresses (catagen phase) and enters a resting phase (telogen). A process similar to morphogenesis occurs each time the telogen hair follicle is induced to re-enter anagen. Specific signals mediating hair follicle induction, regression and reactivation have not yet been identified.
Since substantial proliferative activity is required to produce a mature hair follicle, it is likely that the differential expression of growth factors and their receptors is one important mechanism which controls these processes. A number of growth factors and growth factor receptors, including transforming growth factor beta (TGF-.beta.) and receptors for epidermal growth factor (EGF), have been localized to specific regions of the hair bulb or surrounding stroma at various stages of hair growth, and differential expression of growth factor mRNA for TGF-.beta.1 and TGF-.beta.2 has been detected in developing follicles of embryonic skin (Green et al., J. Invest Dermatol. 83: 118-23 (1984), Heine et al., J. Cell Biol. 105: 2861-76 (1987), Lehnert et al., Development 104: 263-73 (1988) and Pelton et al., Development 106: 759-67 (1989)). Thus, these factors may be involved in hair follicle growth and differentiation.
In mouse skin, the dermis expands and contracts in thickness in parallel with the hair growth cycle ((Sengel, P., Morphogenesis of Skin (Cambridge Univ. Press, Cambridge (1976)). As it grows, the anagen hair follicle migrates through a dermis which is simultaneously expanding to several times its original thickness. This migration of the developing hair follicle through the stromal component during initial formation and in later anagen phases is reminiscent of tumor invasion. On the basis of these observations, it was proposed that collagenases and other proteases may be important in follicle invasion into the deeper dermis (Rogers, et al., J. Invest. Dermatol. 89: 369-79 (1987)), as well as in the remodeling observed during involution of the hair bulb in catagen.
A limiting factor in the analysis of hair follicle growth and development has been the lack of a suitable in vitro model to study these events in a controlled setting. Several cell culture models employing dissociated follicle cells do not support the production of hair specific keratins or other features of follicle differentiation (Imcke et al., J Am. Acad. Dermatol. 17: 779-86 (1987), Jones et al., J. Invest. Dermatol. 90: 58-64 (1988), Limat et al., J. Invest. Dermatol. 87: 485-88 (1986), Noser et al., In vitro Cell Dev. Biol. 23: 541-45 (1987) and Weterings et al., Br. J. Dermatol. 104: 1-5 (1981)). The present inventors have described methods for the enzymatic isolation of intact pelage hair follicles from newborn mouse skin and their maintenance in a 3-dimensional type I collagen gel (Rogers et al., J. Invest. Dermatol. 89: 369-79 (1987)). Such follicles, when reisolated from culture after 7 days, will establish a haired skin when tested in a nude mouse graft model (Rogers et al., supra). When cultured as 3-dimensional organoids, mouse follicles produce a 62 kD cytokeratin band distinct from proteins produced by interfollicular epidermal cells (Rogers et al., supra). The initial studies with this model indicated that follicle cell proliferation was stimulated by epidermal growth factor and cholera toxin, suggesting that this system was useful to analyze responses of follicle cell subpopulations to cytokines. Furthermore, EGF caused follicle cell-mediated lysis of the collagen gel matrix, presumably by stimulating secretion of a collagenase. Thus, this model promised to reveal information regarding the regulation of follicle invasion as well.
The present invention relates to a further analysis of selected growth factors, and pharmaceuticals individually and in combination, with regard to their abilities to stimulate proliferation and collagenase secretion in cultured follicle organoids. The results provide new information which may be relevant to the control of growth and invasion during hair follicle development in vivo.